Use of glp-1 or analogs in treatment of myocardial infarction

ABSTRACT

This invention provides a method of reducing mortality and morbidity after myocardial infarction. GLP-1, a GLP-1 analog, or a GLP-1 derivative, is administered at a dose effective to normalize blood glucose.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/024,980, filed Aug. 30, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method of reducing mortality and morbidityafter myocardial infarction in diabetic patients.

2. Background Information

Morbidity and mortality from cardiovascular disease is higher inpatients with manifest diabetes or impaired glucose tolerance comparedto patients without those disorders. Diabetics account for up to 24% ofthe total number of patients admitted to coronary care units for suspectinfarction, whereas they constitute only about 5% of the generalpopulation [Malmberg and Rydén; Fuller J. H., Diabet. Metab. 19:96-99(1993)]. In-hospital mortality of diabetic patients with myocardialinfarction is twice that of non-diabetics [Hamsten A., et al., J. Int.Med. 736:1-3 (1994) 236 Suppl.; Malmberg K. and Rydén L., Eur. Heart J.9:256-264

(1988)]. Diabetics experience more morbidity and die more often in thepost-acute recovery phase, mostly due to fatal re-infarction andcongestive heart failure [Malmberg and Rydén; Stone P., et al., J. Am.Coll. Cardiol. 14:49-57 (1989); Karlson B. W., et al., Diabet. Med. 10(5):449-54 (1993); Barbash G. I., et al., J. Am. Coll. Cardiol.22:707-713 (1993)]. The difference in mortality and morbidity betweendiabetics and non-diabetics following myocardial infarction persists,despite reduction in the incidence of morbidity and mortality followingacute myocardial infarction [Granger C. B., et al., J. Am. Coll.Cardiol., 21 (4):920-5 (1993); Grines C., et al., N. Engl. J. Med.328:673-679 (1993)].

Factors responsible for the poor prognosis among diabetic patients withacute myocardial infarction may act before, during, or after the acuteevent. They include diffuse coronary atheromatosis, with more advancedand widespread coronary artery disease, which, together with a possiblediabetic cardiomyopathy, may contribute to a high prevalence ofcongestive heart failure. Autonomic neuropathy with impaired painperception and increased resting heart rate variability may also be ofimportance. A coronary thrombus is an essential part of an evolvinginfarction, and notably, platelet activity, coagulation, andfibrinolytic functions have been found to be disturbed in diabeticpatients [Davi G., et al., New England. J. Med., 322:1769-1774 (1990)].

Exaggerated metabolic disturbances in diabetics may play an importantrole. Myocardial infarction causes a reduction in circulating insulin, adramatic increase in adrenergic tone, and the release of stresshormones, such as, cortisone, catecholamines, and glucagon, thattogether enhance hyperglycemia and stimulate lipolysis. The releasedfree fatty acids further injure the myocardium via several mechanisms,and excessive oxidation of free fatty acids may possibly damagenonischemic parts of the myocardium [Rodrigues B., et al.,Cardiovascular Research, 26 (10):913-922 (1992)].

Palliative measures to normalize blood glucose and to control themetabolic cascade that exacerbates infarct damage in diabetics areneeded. In a recent trial, improved metabolic care of diabetic patientsduring acute myocardial infarction, including carefully-monitoredinfusion of insulin and glucose, and post-acute tight regulation ofblood glucose by subcutaneous multidose insulin treatment loweredmortality during the year following myocardial infarction by 30%compared with a control group of diabetics who did not receive insulintreatment unless deemed clinically necessary [Malmberg, K, et al., J.Am. College Cardiology, 26:57-65 (1995)].

Insulin infusion, however, creates the potential for hypoglycemia, whichis defined as blood glucose below 0.3 mM. Hypoglycemia increases therisk of ventricular arrhythmia and is a dangerous consequence of insulininfusion. An algorithm for insulin infusion for diabetics withmyocardial infarction was developed to prevent hypoglycemia [Hendra, T.J., et al., Diabetes Res. Clin. Pract., 16:213-220 (1992)]. However, 21%of the patients developed hypoglycemia under this algorithm. In anotherstudy of glucose control following myocardial infarction, 18% of thepatients developed hypoglycemia when infused with insulin and glucose[Malmberg, K. A., et al., Diabetes Care, 17:1007-1014 (1994)].

Insulin infusion also requires frequent monitoring of blood glucoselevels so that the onset of hypoglycemia can be detected and remedied assoon as possible. In patients receiving insulin infusion in the citedstudy [Malmberg, 1994], blood glucose was measured at least every secondhour, and the rate of infusion adjusted accordingly. Thus, the safetyand efficacy of insulin-glucose infusion therapy for myocardial infarctpatients depends on easy and rapid access to blood glucose data. Such anintense need for monitoring blood glucose places a heavy burden onhealth care professionals, and increases the inconvenience and cost oftreatment. As a result, cardiac intensive care units often do not allotresources for optimizing blood glucose levels in diabetics with acutemyocardial infarction, as might be obtained by intravenousadministration of insulin. Considering the risks and burdens inherent ininsulin infusion, an alternate approach to management of blood glucoseduring acute myocardial infarction in diabetics is needed.

The incretin hormone, glucagon-like peptide 1, abbreviated as GLP-1, isprocessed from proglucagon in the gut and enhances nutrient-inducedinsulin release [Krcymann B., et al., Lancet 2:1300-1303 (1987)].Various truncated forms of GLP-1, are known to stimulate insulinsecretion (insulinotropic action) and cAMP formation [see, e g., Mojsov,S., Int. J. Peptide Protein Research, 40:333-343 (1992)]. A relationshipbetween various in vitro laboratory experiments and mammalian,especially human, insulinotropic responses to exogenous administrationof GLP-1, GLP-1 (7-36) amide, and GLP-1 (7-37) acid has been established[see, e.g., Nauck, M. A., et al., Diabetologia, 36:741-744 (1993);Gutniak, M., et al., New England J. of Medicine, 326 (20):1316-1322(1992); Nauck, M. A., et al., J. Clin. Invest., 91:301-307 (1993); andThorens, B., et al., Diabetes, 42:1219-1225 (1993)]. GLP-1 (7-36) amideexerts a pronounced antidiabetogenic effect in insulin-dependentdiabetics by stimulating insulin sensitivity and by enhancingglucose-induced insulin release at physiological concentrations [GutniakM., et al., New England J. Med. 326:1316-1322 (1992)]. When administeredto non-insulin dependent diabetics, GLP-1 (7-36) amide stimulatesinsulin release, lowers glucagon secretion, inhibits gastric emptyingand enhances glucose utilization [Nauck, 1993; Gutniak, 1992; Nauck,1993].

The use of GLP-1 type molecules for prolonged therapy of diabetes hasbeen obstructed because the serum half-life of such peptides is quiteshort. For example, GLP-1 (7-37) has a serum half-life of only 3 to 5minutes. GLP-1 (7-36) amide has a half-life of about 50 minutes whenadministered subcutaneously. Thus, these GLP molecules must beadministered as a continuous infusion to achieve a prolonged effect[Gutniak M., et al., Diabetes Care 17:1039-1044 (1994)]. In the presentinvention, GLP-1's short half-life and the consequent need forcontinuous administration are not disadvantages because the patient istypically bedridden, in a cardiac intensive care unit, where fluids arecontinuously administered parenterally.

SUMMARY OF THE INVENTION

The present invention provides a method of reducing mortality andmorbidity after myocardial infarction, comprising administering acompound from the group consisting of GLP-1, GLP-1 analogs, GLP-1derivatives, and pharmaceutically-acceptable salts thereof, at a doseeffective to normalize blood glucose, to a patient in need thereof.

The present invention provides the benefits of reduction in mortalityand morbidity after myocardial infarction observed in combined treatmentwith glucose and insulin in diabetics during acute myocardialinfarction, but without the inconvenient and expensive requirement offrequent monitoring of blood glucose, interpretation of blood glucoseresults, and adjustment of insulin dose rate, and without theever-present risk of hypoglycemia that accompanies insulin infusion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of continuous infusion GLP-1 (7-36)amide on average blood glucose concentration (mM)

in five NIDDM patients during the night. The graph also depicts theeffect of continuous insulin infusion on average blood glucoseconcentration

in the same five NIDDM patients, but on a different night.

FIG. 2 is a graph showing the effect of GLP-1 (7-36) amide infusion onaverage blood glucose concentration (mM)

in five NIDDM patients when infused during the day, for three hoursstarting at the beginning of each of three meals. The graph also depictsthe effect of subcutaneous injection of insulin on average blood glucoseconcentration

in the same five NIDDM patients, but on a different day, and withinjection shortly before each meal.

DETAILED DESCRIPTION OF THE INVENTION

“GLP-1” means GLP-1 (7-37). By custom in the art, the amino-terminus ofGLP-1 (7-37) has been assigned number 7 and the carboxy-terminus, number37. The amino acid sequence of GLP-1 (7-37) is well-known in the art,but is presented below for the reader's convenience: (SEQ ID NO:1)NH2-His7-Ala-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20-Glu-Gly-Gln-Ala-Ala25-Lys-Glu-Phe-Ile-Ala30-Trp-Leu-Val-Lys-Gly35-Arg-Gly37-COOH

A “GLP-1 analog” is defined as a molecule having one or more amino acidsubstitutions, deletions, inversions, or additions compared with GLP-1.GLP-1 analogs known in the art include, for example, GLP-1 (7-34) andGLP-1 (7-35), GLP-1 (7-36), Gln9-GLP-1 (7-37), D-Gln9-GLP-1 (7-37),Thr16-Lys18-GLP-1 (7-37), and Ly18-GLP-1 (7-37). Preferred GLP-1 analogsare GLP-1 (7-34) and GLP-1 (7-35), which are disclosed in U.S. Pat. No.:5,118,666, herein incorporated by reference, and also GLP-1 (7-36),which are the biologically processed forms of GLP-1 havinginsulinotropic properties. Other GLP-1 analogs are disclosed in U.S.Pat. No. 5,545,618 which is incorporated herein by reference.

A “GLP-1 derivative” is defined as a molecule having the amino acidsequence of GLP-1 or of a GLP-1 analog, but additionally having chemicalmodification of one or more of its amino acid side groups, α-carbonatoms, terminal amino group, or terminal carboxylic acid group. Achemical modification includes, but is not limited to, adding chemicalmoieties, creating new bonds, and removing chemical moieties.Modifications at amino acid side groups include, without limitation,acylation of lysine ε-amino groups, N-alkylation of arginine, histidine,or lysine, alkylation of glutamic or aspartic carboxylic acid groups,and deamidation of glutamine or asparagine. Modifications of theterminal amino include, without limitation, the desamino, N-lower alkyl,N-di-lower alkyl, and N-acyl modifications. Modifications of theterminal carboxy group include, without limitation, the amide, loweralkyl amide, dialkyl amide, and lower alkyl ester modifications. Loweralkyl is C1-C4 alkyl. Furthermore, one or more side groups, or terminalgroups, may be protected by protective groups known to theordinarily-skilled protein chemist. The α-carbon of an amino acid may bemono- or dimethylated.

A preferred group of GLP-1 analogs and derivatives for use in thepresent invention is composed of molecules of the formula: (SEQ ID NO:2)R1-X-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser- Tyr-Leu20-Y-Gly-Gln-Ala-Ala25-Lys- Z -Phe-Ile- Ala30-Trp-Leu-Val-Lys-Gly35-Arg-R2

and pharmaceutically-acceptable salts thereof, wherein: R1 is selectedfrom the group consisting of L-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;X is selected from the group consisting of Ala, Gly, Val, Thr, Ile, andalpha-methyl-Ala; Y is selected from the group consisting of Glu, Gln,Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu,Gln, Ala, Thr, Ser, and Gly; and R2 is selected from the groupconsisting of NH2, and Gly-OH; provided that the compound has anisoelectric point in the range from about 6.0 to about 9.0 and furtherproviding that when R1 is His, X is Ala, Y is Glu, and Z is Glu, R2 mustbe NH2.

Numerous GLP-1 analogs and derivatives having an isoelectric point inthis range have been disclosed and include, for example: (SEQ ID NO:6)GLP-1 (7-36)NH2 Gly8-GLP-1 (7-36)NH2 Gln9-GLP-1 (7-37) D-Gln9-GLP-1(7-37) acetyl-Lys9-GLP-1 (7-37) Thr9-GLP-1 (7-37) D-Thr9-GLP-1 (7-37)Asn9-GLP-1 (7-37) D-Asn9-GLP-1 (7-37) Ser22-Arg23-Arg24-Gln26-GLP-1(7-37) Thr16-Lys18-GLP-1 (7-37) Lys18-GLP-1 (7-37) Arg23-GLP-1 (7-37)Arg24-GLP-1 (7-37), and the like [see, e.g., WO 91/11457]

Another preferred group of active compounds for use in the presentinvention is disclosed in WO 91/11457, and consists essentially of GLP-1(7-34), GLP-1 (7-35), GLP-1 (7-36), or GLP-1 (7-37), or the amide formthereof, and pharmaceutically-acceptable salts thereof, having at leastone modification selected from the group consisting of:

(a) substitution of glycine, serine, cysteine, threonine, asparagine,glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,phenylalanine, arginine, or D-lysine for lysine at position 26 and/orposition 34; or substitution of glycine, serine, cysteine, threonine,asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine,methionine, phenylalanine, lysine, or a D-arginine for arginine atposition 36;

(b) substitution of an oxidation-resistant amino acid for tryptophan atposition 31;

(c) substitution of at least one of: tyrosine for valine at position 16;lysine for serine at position 18; aspartic acid for glutamic acid atposition 21; serine for glycine at position 22; arginine for glutamineat position 23; arginine for alanine at position 24; and glutamine forlysine at position 26; and

(d) substitution of at least one of: glycine, serine, or cysteine foralanine at position 8; aspartic acid, glycine, serine, cysteine,threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine,leucine, methionine, or phenylalanine for glutamic acid at position 9;serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine,valine, isoleucine, leucine, methionine, or phenylalanine for glycine atposition 10; and glutamic acid for aspartic acid at position 15; and

(e) substitution of glycine, serine, cysteine, threonine, asparagine,glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,or phenylalanine, or the D- or N-acylated or alkylated form of histidinefor histidine at position 7; wherein, in the substitutions is (a), (b),(d), and (e), the substituted amino acids can optionally be in theD-form and the amino acids substituted at position 7 can optionally bein the N-acylated or N-alkylated form.

Because the enzyme, dipeptidyl-peptidase IV (DPP IV), may be responsiblefor the observed rapid in vivo inactivation of administered GLP-1, [see,e.g., Mentlein, R., et al., Eur. J. Biochem., 214:829-835 (1993)],administration of GLP-1 analogs and derivatives that are protected fromthe activity of DPP IV is preferred, and the administration ofGly8-GLP-1 (7-36) NH2, Val8-GLP-1 (7-37) OH, a-methyl-Ala8-GLP-1 (7-36)NH2, and Gly8-Gln21-GLP-1 (7-37) OH, or pharmaceutically-acceptablesalts thereof, is more preferred.

The use in the present invention of a molecule claimed in U.S. Pat. No.5,188,666, which is expressly incorporated by reference, is preferred.Such molecule is selected from the group consisting of a peptide havingthe amino acid sequence: (SEQ ID NO:3)NH2-His7-Ala-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20-Glu-Gly-Gln-Ala-Ala25-Lys-Glu-Phe-Ile-Ala30-Trp-Leu-Val-Xwherein X is selected from the group consisting of Lys and Lys-Gly; anda derivative of said peptide, wherein said peptide is selected from thegroup consisting of: a pharmaceutically-acceptable acid addition salt ofsaid peptide; a pharmaceutically-acceptable carboxylate salt of saidpeptide; a pharmaceutically-acceptable lower alkylester of said peptide;and a pharmaceutically-acceptable amide of said peptide selected fromthe group consisting of amide, lower alkyl amide, and lower dialkylamide.

Another preferred group of molecules for use in the present inventionconsists of compounds, claimed in U.S. Pat. No. 5,512,549, which isexpressly incorporated herein by reference, of the general formula: (SEQID NO:4) R1-Ala-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20-Glu-Gly-Gln-Ala-Ala25-Xaa-Glu-Phe-Ile-Ala30-Trp-Leu-Val-Lys-Gly35-Arg-R3                        |                       R2and pharmaceutically-acceptable salts thereof, wherein R1 is selectedfrom the group consisting of 4-imidazopropionyl, 4-imidazoacetyl, or4-imidazo-α, α dimethyl-acetyl; R2 is selected from the group consistingof C6-C10 unbranched acyl, or is absent; R3 is selected from the groupconsisting of Gly-OH or NH2; and, Xaa is Lys or Arg, may be used inpresent invention.

More preferred compounds of SEQ ID NO:4 for use in the present inventionare those in which Xaa is Arg and R2 is C6-C10 unbranched acyl.

Highly preferred compounds of SEQ ID NO:4 for use in the presentinvention are those in which Xaa is Arg, R2 is C6-C10 unbranched acyl,and R3 is Gly-OH.

More highly preferred compounds of SEQ ID NO:4 for use in the presentinvention are those in which Xaa is Arg, R2 is C6-C10 unbranched acyl,R3 is Gly-OH, and R1 is 4-imidazopropionyl.

The most preferred compound of SEQ ID NO:4 for use in the presentinvention is that in which Xaa is Arg, R2 is C8 unbranched acyl, R3 isGly-OH, and R1 is 4-imidazopropionyl.

The use in the present invention of a molecule claimed in U.S. Pat. No.5,120,712, which is expressly incorporated by reference, is highlypreferred. Such molecule is selected from the group consisting of apeptide having the amino acid sequence: (SEQ ID NO:1)NH2-His7-Ala-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20-Glu-Gly-Gln-Ala-Ala25-Lys-Glu-Phe-Ile-Ala30-Trp-Leu-Val-Lys-Gly35-Arg-Gly37-COOHand a derivative of said peptide, wherein said peptide is selected fromthe group consisting of: a pharmaceutically-acceptable acid additionsalt of said peptide; a pharmaceutically-acceptable carboxylate salt ofsaid peptide; a pharmaceutically-acceptable lower alkylester of saidpeptide; and a pharmaceutically-acceptable amide of said peptideselected from the group consisting of amide, lower alkyl amide, andlower dialkyl amide.

The use of GLP-1 (7-36) amide, or a pharmaceutically-acceptable saltthereof, in the present invention is most highly preferred. The aminoacid sequence of GLP-1 (7-36) amide is: (SEQ ID NO:5)NH2-His7-Ala-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20-Glu-Gly-Gln-Ala-Ala25-Lys-Glu-Phe-Ile-Ala30-Trp-Leu-Val-Lys-Gly35-Arg-NH2

Methods for preparing the active compound used in the present invention,namely, GLP-1, an GLP-1 analog, or a GLP-1 derivative used in thepresent invention are well-known, and are described in U.S. Pat. Nos.5,118,666, 5,120,712, and 5,523,549, which are incorporated byreference.

The amino acid portion of the active compound used in the presentinvention, or a precursor thereto, is made either by 1) solid-phasesynthetic chemistry; 2) purification of GLP molecules from naturalsources; or 3) recombinant DNA technology.

Solid phase chemical synthesis of polypeptides is well known in the artand may be found in general texts in the area such as Dugas, H. andPenney, C., Bioorganic Chemistry, Springer-Verlag, New York (1981), pp.54-92, Merrifield, J. M., Chem. Soc., 85:2149 (1962), and Stewart andYoung, Solid Phase Peptide Synthesis, Freeman, San Francisco (1969) pp.24-66.

For example, the amino acid portion may be synthesized by solid-phasemethodology utilizing a 430A peptide synthesizer (PE-Applied Biosystems,Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404) and synthesiscycles supplied by PE-Applied Biosystems. BOC-amino acids and otherreagents are commercially available from PE-Applied Biosystems and otherchemical supply houses. Sequential Boc chemistry using double coupleprotocols are applied to the starting p-methyl benzhydryl amine resinsfor the production of C-terminal carboxamides. For the production ofC-terminal acids, the corresponding PAM resin is used. Asn, Gln, and Argare coupled using preformed hydroxy benzotriazole esters. The followingside chain protecting groups may be used:

Arg, Tosyl

Asp, cyclohexyl

Glu, cyclohexyl

Ser, Benzyl

Thr, Benzyl

Tyr, 4-bromo carbobenzoxy

Boc deprotection may be accomplished with trifluoroacetic acid inmethylene chloride. Following completion of the synthesis the peptidesmay be deprotected and cleaved from the resin with anhydrous hydrogenfluoride (HF) containing 10% meta-cresol. Cleavage of the side chainprotecting group(s) and of the peptide from the resin is carried out at−5° C. to 5° C., preferably on ice for 60 minutes. After removal of theHF, the peptide/resin is washed with ether, and the peptide extractedwith glacial acetic acid and lyophilized.

Techniques well-known to the ordinarily-skilled artisan in recombinantDNA technology may be used to prepare the active compound used inpresent invention. In fact, recombinant DNA methods may be preferablebecause of higher yield. The basic steps in recombinant production are:

-   -   a) isolating a natural DNA sequence encoding a GLP-1 molecule or        constructing a synthetic or semi-synthetic DNA coding sequence        for a GLP-1 molecule,    -   b) placing the coding sequence into an expression vector in a        manner suitable for expressing proteins either alone or as a        fusion proteins,    -   c) transforming an appropriate eukaryotic or prokaryotic host        cell with the expression vector,    -   d) culturing the transformed host cell under conditions that        will permit expression of a GLP-1 molecule, and    -   e) recovering and purifying the recombinantly produced GLP-1        molecule.

As previously stated, the coding sequences may be wholly synthetic orthe result of modifications to the larger, native glucagon-encoding DNA.A DNA sequence that encodes preproglucagon is presented in Lund, et al.,Proc. Natl. Acad. Sci. U.S.A. 79:345-349 (1982) and may be used asstarting material in the semisynthetic production of the compounds ofthe present invention by altering the native sequence to achieve thedesired results.

Synthetic genes, the in vitro or in vivo transcription and translationof which results in the production of a GLP-1 molecule, may beconstructed by techniques well known in the art. Owing to the naturaldegeneracy of the genetic code, the skilled artisan will recognize thata sizable yet definite number of DNA sequences may be constructed, allof which encode GLP-1 molecules.

The methodology of synthetic gene construction is well-known in the art.See Brown, et al. (1979) Methods in Enzymology, Academic Press, N.Y.,Vol. 68, pgs. 109-151. The DNA sequence is designed from the desiredamino acid sequence using the genetic code, which is easily ascertainedby the ordinarily-skilled biologist. Once designed, the sequence itselfmay be generated using conventional DNA synthesizing apparatus such asthe Model 380A or 380B DNA synthesizers (PE-Applied Biosystems, Inc.,850 Lincoln Center Drive, Foster City, Calif. 94404).

To express the amino acid portion of a compound used in the presentinvention, one inserts the engineered synthetic DNA sequence in any oneof many appropriate recombinant DNA expression vectors through the useof appropriate restriction endonucleases. See generally Maniatis et al.(1989) Molecular Cloning; A Laboratory Manual, Cold Springs HarborLaboratory Press, N.Y., Vol. 1-3. Restriction endonuclease cleavagesites are engineered into either end of the GLP-1 molecule-encoding DNAto facilitate isolation from, and integration into, amplification andexpression vectors well-known in the art. The particular endonucleasesemployed will be dictated by the restriction endonuclease cleavagepattern of the parent expression vector employed. Restriction sites arechosen to properly orient the coding sequence with control sequences,thereby achieving proper in-frame reading and expression of the proteinof interest. The coding sequence must be positioned to be in properreading frame with the promoter and ribosome binding site of theexpression vector, both of which are functional in the host cell inwhich the protein is to be expressed.

To achieve efficient transcription of the synthetic gene, it must beoperably associated with a promoter-operator region. Therefore, thepromoter-operator region of the synthetic gene is placed in the samesequential orientation with respect to the ATG start codon of thesynthetic gene.

A variety of expression vectors useful for transforming prokaryotic andeukaryotic cells are well known in the art. See The Promega BiologicalResearch Products Catalogue (1992) (Promega Corp., 2800 Woods HollowRoad, Madison, Wis., 53711-5399); and The Stratagene Cloning SystemsCatalogue (1992) (Stratagene Corp., 11011 North Torrey Pines Road, LaJolla, Calif., 92037). Also, U.S. Pat. No. 4,710,473 describes circularDNA plasmid transformation vectors useful for expression of exogenousgenes in E. coli at high levels. These plasmids are useful astransformation vectors in recombinant DNA procedures and

-   -   (a) confer on the plasmid the capacity for autonomous        replication in a host cell;    -   (b) control autonomous plasmid replication in relation to the        temperature at which host cell cultures are maintained;    -   (c) stabilize maintenance of the plasmid in host cell        populations;    -   (d) direct synthesis of a protein product indicative of plasmid        maintenance in a host cell population;    -   (e) provide in-series restriction endonuclease recognition sites        unique to the plasmid; and    -   (f) terminate mRNA transcription.        These circular DNA plasmids are useful as vectors in recombinant        DNA procedures for securing high levels of expression of        exogenous genes.

Having constructed an expression vector for the amino acid portion of acompound used in the present invention, the next step is to place thevector into a suitable cell and thereby construct a recombinant hostcell useful for expressing the polypeptide. Techniques for transformingcells with recombinant DNA vectors are well known in the art and may befound in such general references as Maniatis, et al. supra. Host cellsmade be constructed from either eukaryotic or prokaryotic cells.

Prokaryotic host cells generally produce the protein at higher rates andare easier to culture. Proteins expressed in high-level bacterialexpression systems characteristically aggregate in granules or inclusionbodies, which contain high levels of the overexpressed protein. Suchprotein aggregates typically must be recovered, solubilized, denaturedand refolded using techniques well known in the art. See Kreuger, et al.(1990) in Protein Folding, Gierasch and King, eds., pgs 136-142,American Association for the Advancement of Science Publication No.89-18S, Washington, D.C.; and U.S. Pat. No. 4,923,967.

Alterations to a precursor GLP-1 or GLP-1 analog amino acid sequence, toproduce a desired GLP-1 analog or GLP-1 derivative, are made bywell-known methods: chemical modification, enzymatic modification, or acombination of chemical and enzymatic modification of GLP-1 precursors.The techniques of classical solution phase methods and semi-syntheticmethods may also be useful for preparing the GLP-1 molecules used in thepresent invention. Methods for preparing the GLP-1 molecules of thepresent invention are well known to an ordinarily skilled peptidechemist.

Addition of an acyl group to the epsilon amino group of Lys34 may beaccomplished using any one of a variety of methods known in the art. SeeBioconjugate Chem. “Chemical Modifications of Proteins: History andApplications” pages 1, 2-12 (1990) and Hashimoto et al., PharmaceuticalRes. 6(2):171-176 (1989).

For example, an N-hydroxy-succinimide ester of octanoic acid can beadded to the lysyl-epsilon amine using 50% acetonitrile in boratebuffer. The peptide can be acylated either before or after theimidazolic group is added. Moreover, if the peptide is preparedrecombinantly, acylation prior to enzymatic cleavage is possible. Also,the lysine in the GLP-1 derivative can be acylated as taught inWO96-29342, which is incorporated herein by reference.

The existence and preparation of a multitude of protected, unprotected,and partially-protected, natural and unnatural, functional analogs andderivatives of GLP-1 (7-36)amide and GLP-1 (7-37) molecules have beendescribed in the art [see, e.g., U.S. Pat. Nos. 5,120,712 and 5,118,666,which are herein incorporated by reference, and Orskov, C., et al., J.Biol. Chem., 264(22):12826-12829 (1989) and WO 91/11457 (Buckley, D. I.,et al., published Aug. 8, 1991)].

Optionally, the amino and carboxy terminal amino acid residues of GLP-1derivatives may be protected, or, optionally, only one of the termini isprotected. Reactions for the formation and removal of such protectinggroups are described in standard works including, for example,“Protective Groups in Organic Chemistry”, Plenum Press, London and NewYork (1973); Green, T. H., “Protective Groups in Organic Synthesis”,Wiley, New York (1981); and “The Peptides”, Vol. I, Schröder and Lübke,Academic Press London and New York (1965). Representativeamino-protecting groups include, for example, formyl, acetyl, isopropyl,butoxycarbonyl, fluorenylmethoxycarbonyl, carbobenzyloxy, and the like.Representative carboxy-protecting groups include, for example, benzylester, methyl ester, ethyl ester, t-butyl ester, p-nitro phenyl ester,and the like.

Carboxy-terminal, lower-alkyl-ester, GLP-1 derivatives used in thepresent invention are prepared by reacting the desired (C1-C4) alkanolwith the desired polypeptide in the presence of a catalytic acid such ashydrochloric acid. Appropriate conditions for such alkyl ester formationinclude a reaction temperature of about 50° C. and reaction time ofabout 1 hour to about 3 hours. Similarly, alkyl ester derivatives of theAsp and/or Glu residues can be formed.

Preparation of a carboxamide derivative of a compound used in thepresent invention is formed, for example, as described in Stewart, J.M., et al., Solid Phase Peptide Synthesis, Pierce Chemical CompanyPress, 1984.

A pharmaceutically-acceptable salt form of GLP-1, of a GLP-1 analog, orof a GLP-1 derivative may be used in the present invention. Acidscommonly employed to form acid addition salts are inorganic acids suchas hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like. Preferred acid addition salts are those formedwith mineral acids such as hydrochloric acid and hydrobromic acid, and,especially, hydrochloric acid.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like. The salt formsare particularly preferred.

A GLP-1, GLP-1 analog, or GLP-1 derivative used in the present inventionmay be formulated with one or more excipients before use in the presentinvention. For example, the active compound used in the presentinvention may be complexed with a divalent metal cation by well-knownmethods. Such metal cations include, for example, Zn++, Mn++, Fe++,Co++, Cd++, Ni++, and the like.

Optionally, the active compound used in the present invention may becombined with a pharmaceutically-acceptable buffer, and the pH adjustedto provide acceptable stability, and a pH acceptable for parenteraladministration.

Optionally, one or more pharmaceutically-acceptable anti-microbialagents may be added. Meta-cresol and phenol are preferredpharmaceutically-acceptable anti-microbial agents. One or morepharmaceutically-acceptable salts may be added to adjust the ionicstrength or tonicity. One or more excipients may be added to furtheradjust the isotonicity of the formulation. Glycerin is an example of anisotonity-adjusting excipient.

Administration may be via any route known to be effective by thephysician of ordinary skill. Parenteral administration is preferred.Parenteral administration is commonly understood in the medicalliterature as the injection of a dosage form into the body by a sterilesyringe or some other mechanical device such as an infusion pump.Parenteral routes include intravenous, intramuscular, subcutaneous,intraperitoneal, intraspinal, intrathecal, inracerebroventricular,intraarterial, subarachnoid, and epidural. Intravenous, intramuscular,and subcutaneous routes of administration of the compounds used in thepresent invention are more preferred. Intravenous and subcutaneousroutes of administration of the compounds used in the present inventionare yet more highly preferred. For parenteral administration, an activecompound used in the present invention preferably is combined withdistilled water at an appropriate pH.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the active compound used in thepresent invention. Extended duration may be obtained by selectingappropriate macromolecules, for example, polyesters, polyamino acids,polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose,carboxymethylcellulose, or protamine sulfate, and by selecting theconcentration of macromolecules, as well as the methods ofincorporation, in order to prolong release. Another possible method toextend the duration of action by controlled release preparations is toincorporate an active compound used in the present invention intoparticles of a polymeric material such as polyesters, polyamino acids,hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating a compound into these polymericparticles, it is possible to entrap a compound used in the presentinvention in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules, respectively, or incolloidal drug delivery systems, for example, liposomes, albuminmicrospheres, microemulsions, nanoparticles, and nanocapsules, or inmacroemulsions. Such teachings are disclosed in Remington'sPharmaceutical Sciences (1980).

A diagnosis of “myocardial infarction” is one involving medicaljudgment, and typically relies a finding of at least two of thefollowing symptoms and indications:

-   -   1) chest pain of at least 15 minute duration;    -   2) at least two values of serum creatine kinase and serum        creatine kinase B at least two standard deviations above the        normal range 10-16 h after onset of symptoms;    -   3) two or more serum lactate dehydrogenase levels that are at        least two standard deviations above the normal range within        48-72 hours after onset of symptoms, including an isoenzyme        pattern typical of myocardial infarction; and    -   4) development of new Q waves and/or initial ST elevation        followed by T-wave inversion in at least two of the 12 standard        ECG leads.

The acute phase of myocardial infarction occurs during the first 72hours after the onset of the symptoms or indications described above.The treatment which is the subject of this invention is given during theacute phase of myocardial infarction, that is, in acute myocardialinfarction.

A patient in need of the compounds used in the present invention is onewho is in the acute phase of myocardial infarction, and who also isincapable of auto-regulation of blood glucose. A patient is incapable ofauto-regulation if that patient: 1) was previously diagnosed withinsulin-dependent diabetes (IDDM) or non-insulin dependent diabetes(NIDDM), according to the definitions of the National Diabetes DataGroup [Diabetes, 28:1039-1057 (1979)]; 2) has a blood glucose levelgreater than 11 mmol/liter, even without a previous diagnosis ofdiabetes; or 3) has an abnormal glucose tolerance.

The dose of GLP-1, GLP-1 analog, or GLP-1 derivative effective tonormalize a patient's blood glucose level will depend on a number offactors, among which are included, without limitation, the patient'ssex, weight and age, the severity of inability to regulate bloodglucose, the underlying causes of inability to regulate blood glucose,whether glucose, or another carbohydrate source, is simultaneouslyadministered, the route of administration and bioavailability, thepersistence in the body, the formulation, and the potency. Whereadministration is continuous, a suitable dosage rate is between 0.25 and6 pmol/kg body weight/min, perferably from about 0.5 to about 1.2pmol/kg/min. Where administration is intermittent, the dose peradministration should take into account the interval between doses, thebioavailability of GLP-1, GLP-1 analog, or GLP-1 derivative, and thelevel needed to effect normal blood glucose. It is within the skill ofthe ordinary physician to titrate the dose and rate of administration ofGLP-1, GLP-1 analog, or GLP-1 derivative to achieve the desired clinicalresult.

The present invention will be more readily understood by reference tospecific examples, which are provided to illustrate, not to limit, thepresent invention.

EXAMPLE 1

GLP-1 (7-36) amide was administered by a subcutaneous infusion at a doserate of 1.2 pmol/kg/hr, for ten hours during the night, to five patientshaving non-insulin dependent diabetes (NIDDM). As a control, insulin wascontinuously infused in the same five patients, but on a different daythan the GLP-1 (7-36) amide infusion. The rate of insulin infusion wasadjusted every two hours to achieve optimum control, and to avoidhypoglycemia. As demonstrated by the data in Table 1, and in FIG. 1,subcutaneous infusion of GLP-1 (7-36) amide nearly normalized bloodglucose without inducing hypoglycemia in any of the patients. Themetabolic control with GLP-1 (7-36) amide was better than that achievedby insulin, and the average blood glucose level was lower for GLP-1(7-36) amide treatment than for the control by a statisticallysignificant amount at 23:00, 0:00, and at 1:00. TABLE 1 Average bloodglucose levels for five NIDDM patients continuously infused for tenhours during the night with GLP-1 (7-36) amide. In a control study withthe same patients on a different day, insulin was administered bycontinuous infusion. Insulin Infusion GLP-1 Infusion (Control) AverageAverage Blood Blood Glucose Std. Error Glucose Std. Error Hour (mM) (mM)(mM) (mM) 21:00  7.5 0.45 6.9 0.68 22:00  5.4 0.76 6.6 0.55 23:00  4.10.16 5.9 0.98 0:00 4.4 0.23 5.6 0.90 1:00 4.4 0.29 5.1 0.58 2:00 4.80.34 5.2 0.58 3:00 5.2 0.41 5.4 0.30 4:00 5.4 0.41 5.7 0.25 5:00 5.80.41 6.0 0.30 6:00 6.0 0.45 6.1 0.38 7:00 6.2 0.45 6.1 0.33

EXAMPLE 2

During the day, GLP-1 (7-36) amide was infused into five NIDDM patientsfor three hours during breakfast, lunch, and dinner. The infusion timeswere 7:30-10:30 (breakfast), 10:30-1:30 (lunch), and 4:30-7:30 (dinner),as indicated in FIG. 2. In a control experiment in the same five NIDDMpatients conducted on a different day, insulin was injectedsubcutaneously just before the start of the meals, as indicated in FIG.2. While GLP-1 was infused, the post-prandial glucose excursionsobserved with insulin injection were eliminated, and normal bloodglucose levels were maintained. Immediately after terminating each GLP-1(7-36) amide infusion, the blood glucose level increased significantly.No untoward side effects of GLP-1 (7-36) amide were observed. These dataindicate that GLP-1 (7-36) amide infusion more effectively controlspost-prandial glucose levels than insulin injection, and that thecontrol is effective as long as GLP-1 (7-36) amide infusion iscontinued. TABLE 2 Average blood glucose levels for five NIDDM patientsinfused with GLP-1 (7-36) amide for three hours, beginning at the startof each meal. In a control study with the same patients on a differentday, insulin was administered by subcutaneous injection just before eachmeal. Meals began at 7:30, 10:30, and at 4:30. Insulin GLP-1Subcutaneous Infusion Injection Average Average Blood Std. Blood Std.Glucose Error Glucose Error Hour (mM) (mM) (mM) (mM)  7:00 5.4 0.35 6.10.41  8:00 4.9 0.38 7.0 0.51  9:00 5.7 0.59 9.1 0.74 10:00 5.8 1.06 9.90.78 11:00 8.1 0.94 8.2 0.76 12:00 9.4 0.59 6.5 0.74 13:00 7.2 1.18 9.10.90 14:00 5.3 1.21 8.1 0.91 15:00 7.2 0.71 7.0 0.87 16:00 10.4  0.267.2 0.57 17:00 9.2 1.06 6.5 0.59 18:00 5.7 1.59 7.3 0.65 19:00 6.6 0.946.1 0.59 20:00 8.3 0.71 6.0 0.41 21:00 9.3 0.71 6.4 0.44

1-12. (canceled)
 13. A method for preventing or ameliorating injury to the myocardium following a myocardial infarction wherein an initial injury to the myocardium has occurred due to the myocardial infarction comprising administering to an individual an effective amount of a composition comprising GLP-1, a GLP-1 analog, or a GLP-1 derivative in a pharmaceutical carrier.
 14. The method of claim 13 wherein the injury to the myocardium is caused by free fatty acid oxidation.
 15. The method of claim 13 wherein the individual has hyperglycemia.
 16. The method of claim 13 wherein the individual has abnormal glucose tolerance.
 17. The method of claim 13 wherein the GLP-1, GLP-1 analog, or GLP-1 derivative is administered during the acute phase of a myocardial infarction.
 18. A method of controlling the metabolic cascade that exacerbates infarct damage comprising administering GLP-1, a GLP-1 analog, or a GLP-1 derivative to a patient in need thereof.
 19. The method of claim 18 wherein the patient has suffered a myocardial infarction.
 20. The method of claim 19 wherein the GLP-1, GLP-1 analog, or GLP-1 derivative is administered during the acute phase of a myocardial infarction.
 21. The method of claim 18 wherein the infarct damage occurs following a period of ischemia.
 22. The method of claim 18 wherein the patient has hyperglycemia.
 23. The method of claim 22 wherein the hyperglycemia is caused by a stress response to a myocardial infarction.
 24. The method of claim 18 wherein the patient has abnormal glucose tolerance.
 25. The method of claim 24 wherein the abnormal glucose tolerance is caused by a stress response to a myocardial infarction.
 26. A method for preventing or ameliorating damage to non-ischemic myocardium following a myocardial infarction comprising administering GLP-1, a GLP-1 analog, or GLP-1 derivative to a patient in need thereof.
 27. The method of claim 26 wherein the GLP-1, GLP-1 analog, or GLP-1 derivative is administered during the acute phase of a myocardial infarction.
 28. The method of claim 26 wherein the patient has hyperglycemia.
 29. The method of claim 28 wherein the hyperglycemia is caused by a stress response to a myocardial infarction.
 30. The method of claim 26 wherein the patient has abnormal glucose tolerance.
 31. The method of claim 30 wherein the abnormal glucose tolerance is caused by a stress response to a myocardial infarction. 